Safe vaccine for hepatitis containing polymerized serum albumin

ABSTRACT

Methods and compositions are provided for an inexpensive safe vaccine for hepatitis infection. Immunogenic polymerized human albumin free of other hepatitis related immunogens is employed in a physiologically acceptable carrier as a vaccine for protection against hepatitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 299,452, filed Sept. 4,1981 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Hepatitis is a viral disease which can exist in both infectious andnon-infectious form. It is of particular concern because of itstransmission through blood transfusions. It is known that there are manycarriers of the virus which are not subject to the symptoms of thedisease. Nevertheless, these people are able to transmit the disease toothers who are susceptible to infection, particularly through bloodtransfusions, where the blood is not carefully monitored.

Because of the widespread character of hepatitis, it would be desirableto be able to vaccinate people for the disease. For the most part,vaccines have relied upon the protein coat of the virus, which is onlydifficultly attainable and must be carefully purified to avoid anyinclusion of the viral chromosome. Because of the expensive nature ofthe isolation and purification of the protein coat, an inexpensivevaccine has not been available which could be used, particularly inthose areas which are unable to afford the high cost of the presentlyavailable vaccines.

2. Description of the Prior Art

Human serum albumin and the hepatitis B surface antigen are associatedwith the viral coat protein of the hepatitis B virus. Neurath et al.(1974) PNAS USA 71:2663; Ionescu-Matiu et al. (1980) J. Med. Virol.6:175; Tiollais et al. (1981) Science 213:406. Thus, common occurrenceof albumin molecules in the coat protein of viruses replicating in theliver is naturally expected. In vitro aging or heat or glutaraldehydepolymerization of albumin results in a product which elicits antibodiesin immunized animals. Onica et al. (1980) Mol. Immunol. 17, 783.Antibodies to polymerized albumin have also been encountered in the seraof patients with acute or chronic liver disease. Lenkei et al. (1977) J.Med. Virol. 1, 29. Physiological and pathological production ofantibodies to polymerized human albumin (PHALB) have been studied byOnica and Lenkei (Onica et al (1978) Immunochemistry 15, 941; Lenkei andGhetie (1977) J. Immunol. Methods 16, 23 and Imai et al. (1979)Gastroenterology 76, 242. The interaction of PHALB with Clq and therelationship of serologic reactivity with PHALB in sera from patientswith and without liver disease have been reported be Milich et al.(1980) Gastroenterology 79, 1116, and Milich et al (1981)Gastroenterology 81, 218.

SUMMARY OF THE INVENTION

A safe inexpensive vaccine is provided by employing polymerized humanalbumin in a physiologically acceptable carrier as a vaccine forhepatitis in the absence of other hepatitis virus derived immunogens. Bypolymerizing human albumin to provide at least a hexamer, an activeimmunogen is produced which upon injection produces an immunogenicresponse to PHALB, but not to monomeric albumin, affording the hostprotection from hepatitis.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Polymerized human serum albumin (PHALB) is prepared and employed in aphysiologically acceptable carrier as a vaccine for hepatitis virus. Thepolymerized albumin composition is desirably on the average at least ahexamer, preferably about an octamer and should generally be less thanabout a dodecamer, preferably less than about a decamer (6-12; 7-10),wherein 90 or greater weight percent is of the same degree ofpolymerization, more preferably having the average composition of anoctamer. The polymerized human albumin can be formed in any conventionalway, by aging, using heat or light, by chemical cross linking, forexample, with aldehydes e.g. formaldehyde, dialdehydes, e.g.glutaraldehyde, or other physiologically acceptable tanning agents.Conditions can be chosen so as to optimize the formation of the desireddegree of polymerization, followed by purification, if desired.Sedimentation or centrifugation may be employed for large scaleseparation by molecular weight, employing isopycnic banding, gradientdensity chromatography centrifugation, or molecular seiving by gelchromatography in large columns. The polymerized human albumin may thenbe chemically or thermally aggregated using physiologically acceptablemultivalent cations or heat.

While the polymerized albumin has been indicated for humans, polymerizedalbumin allogeneic for any species can be used for the mammalian speciesto provide an inexpensive vaccine against hepatitis. Therefore, fordomestic animals or other mammals which may be subject to hepatitisinfection, the subject invention can be used as a vaccine.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a dead vaccine are applicable. Theseinclude oral application on a solid physiologically acceptable base orin a physiologically acceptable dispersion, parenterally, by injectionor the like. The dosage of the vaccine will depend on the route ofadministration and will vary according to the size of the host. Becausethe vaccine has few if any side effects, relatively large dosages may beused without injury to the host. Normally, the amount of the vaccinewill be from about 1 μg to 20.0 mg per kilogram of host, more usuallyfrom about 5 μg to 2.0 mg given subcutaneously or intramuscularly aftermixing with an appropriate carrier or an adjuvant to enhanceimmunization with the vaccine.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminium hydroxide or phosphate (alum), commonlyused as 0.05 to 0.1 percent solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol) used as 0.25percent solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between 70° to 101° C. for 30 to 2minute periods respectively, aggregation by reacting with pepsin treated(Fab) antibodies to albumin, mixture with bacterial cells such as C.parvum or endotoxins or lipopolysaccharide components of gram-negativebacteria, emulsion in physiologically acceptable oil vehicles such asmannide mono-oleate (Aracel A) or emulsion with 20 percent solution of aperfluorocarbon (Fluosol-DA) used as a blood substitute. More novelmethods of adjuvanticity would include bacterial toxins against whichthe host has been preimmunized, for example by coupling 5 moles of thevaccine per mole of tetanus toxoid or diptheria toxoid, the compoundvaccine could elicit enhanced immune response to PHALB. The amount ofthe adjuvant will vary widely depending upon the nature of the adjuvant,generally varying from 0.1 to 100 times the weight of the immnogen, moreusually from 1 to 10 times.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for PHALB. The assays may be performed by labelling PHALBwith conventional labels, such as radionuclides, enzymes, flourescers,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932, 4,174,384, and3,949,064, as illustrative of these types of assays. The types of assaysmay be divided between homogeneous, which do not involve a separationstep, and heterogeneous, which do involve a separation step.

Radioimmunoassays are illustrative of the heterogeneous assays. Aradioimmunoassay could be performed by binding PHALB to a surface,either a particle or the surface of a container, adding the serum sampleto the bound PHALB, and allowing the mixture to incubate for sufficienttime for any antibody to PHALB to react with the bound PHALB. One wouldthen add radionuclide-labelled PHALB to the container, incubate for aperiod of time sufficient for the labelled PHALB to bind any antibodybound to the surface, wash, and then measure the radioactivity bound tothe surface. Alternative protocols may also be employed.

In a homogeneous assay, PHALB could be substituted with fluorescermolecules, the labelled PHALB combined with the sample, followed byaddition of antibodies to the fluorescer. Depending upon the choice ofthe fluorescer, binding of the antibody to the fluorescer could resultin an increase or decrease in fluorescence. The binding of antibodies tothe PHALB will inhibit binding of antibodies to the fluorescer, so thatby measuring the fluoresence of the assay medium in comparison with anassay medium having known amounts of antibodies to PHALB, the presenceof antibodies to PHALB in the sample could be determined. The particularmanner in which the presence of the antibodies to the PHALB in a serumsample is determined is not a significant aspect of this invention.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

To prepare cross-linked PHALB, 120 mg of crystalline human serum albumin(HSA) is dissolved in 3.6 ml of 0.1M phosphate buffer (pH 6.8).Polymerization is accomplished by the addition of 0.4 ml of a 2.5%solution of glutaraldehyde solution in 0.1M phosphate buffer (pH 6.8).After two hours at room temperature, the reaction mixture is dialyzedagainst 3×500 ml PBS, changed at 3, 6 and 18 hour intervals. The PHALBis then treated with 0.1% sodium borohydride to stabilize the polymerand neutralize unreacted aldehyde groups and then chromatographedthrough Sephacryl S-300 gel column in PBS. The first major peak isseparated and determined to have an average molecular weight of 500,000daltons.

For the performance of radioimmunoassay, HSA is prelabelled with ¹²⁵ Iusing the chloramine-T or the Hunter-Bolten reagent and then subjectedto the above polymerization procedure to obtain ¹²⁵ I-PHALB probe.Polystyrene beads of 0.5 cm diameter are first coated with a 0.5 mg/mlsolution of PHALB in PBS. The PHALB-coated beads are washed with PBScontaining 0.2% between 20 and 0.5% BSA (pH 6.4) and air dried. Testsera are serially diluted with PBS and 2.5% BSA (pH 7.2) for titrationand to each dilution a PHALB-coated bead is added, incubated for 2 hoursat 37° C. and then the beads are washed four times with deionized water.Then each bead is reacted with ¹²⁵ I-labelled PHALB probe, incubated for4 hours at 37° C., washed 4X and the radioactivity bound to the bead iscounted in a gamma counter. The highest dilution of the serum samplegiving counts of more than 2.1 times the negative control is consideredpositive and the end-point of titration. The negative control beads arecoated with polymerized transferrin.

Several human sera from patients with acute hepatitis A, sera fromconvalescent hepatitis B and sera from chronic nonA/nonB hepatitisreveal the presence of antibodies to PHALB in the gamma globulinfraction. The serologic specificity of the antibodies is established byneutralization reactions using 4 units of limiting antibodies. Both 0.1μg of PHALB and purified 3.0 μg of hepatitis virus envelope proteinneutralize the antibodies, whereas up to 1000 μg of HSA and other plasmaproteins fail to inhibit the human antibodies to PHALB. Thus, it isapparent that purified envelope protein of HBV and PHALB share theserologic determinant and indicate that antibodies to PHALB wouldprovide an effective protection against infection by varioushepatotropic viruses including the hepatitis B virus.

For preparation of the vaccine, 6.0 mg of PHALB is mixed with 20 mg ofmannitol per ml of PBS and heated at 60° C. for 11 hours, followed byheating at 101° C. for 1-2 minutes. The heat-treated PHALB is mixed withequal volume of 0.1% aluminium hydroxide and tested for toxicity andpyrogenicity. The PHALB may then be used as a vaccine for immunizationand formulated in the conventional ways. The mineral ion (aluminium) isrequired for induction of primary immune response, however, because itforms prolonged granuloma at the site of immunization, for the boosterthe heat-treated PHALB alone, without the adjuvanticity of alum, may beused for secondary immunization.

Effective immunization can be monitored by serologic tests of host serumfor antibodies to PHALB. The primary immune response of IgM-type isdistinguished from the IgG-type secondary immune response by treatmentof the test serum with 0.1M 2-mercaptoethanol. The secondary immuneresponse to PHALB is long lasting and protective against infection withhepatotropic viruses.

In accordance with the subject invention, an inexpensive safe vaccine isprovided which can be repeatedly administered to mammalian hosts toprovide for a rapid immune response in the event of hepatitis infectionby any of the hepatotrophic viruses. Human serum albumin can be readilyobtained in pure form and then polymerized to physiologicalcompositions. Thus, populations which cannot afford vaccines prepared bymore expensive techniques can have protection from hepatitis.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for protecting a host susceptible tohepatitis infection which comprises:administering to said host aneffective amount of a vaccine consisting essentially of polymerisedserum albumin as the immunogen to elicit an immunogenic response in aphysiologically acceptable carrier to said host, said polymerized serumalbumin having from six to twelve serum albumin units, and being free ofany other portions of hepatotropic viruses.
 2. A method according toclaim 1, wherein said administering is done at least twice to the samehost.
 3. A method according to claims 1 or 2, wherein said polymerizedserum albumin is on the average an octamer.
 4. A method according to anyof claims 1 or 2, wherein said polymerized serum albumin is thermally orchemically aggregated.
 5. A vaccine useful for vaccination for amammalian host against hepatitis which comprises polymerized serumalbumin in combination with an immunogenic adjuvant, wherein saidpolymerized serum albumin has about six to twelve units, is free ofother proteins or genetic material derived from hepatotropic viruses,and is in an amount sufficient to elicit an immunogenic response.
 6. Avaccine according to claim 5, wherein said polymerized serum albumin isthermally or chemically aggregated.